Rapid native single cell mass spectrometry

ABSTRACT

A method for analyzing single cells by mass spectrometry includes the steps of providing a plurality of cells in a liquid medium and placing the cells and liquid medium in a single cell isolation and ejection system. Liquid medium containing a single cell is released from the single cell isolation and ejection system. The liquid medium and single cell are captured in a capture probe containing a flowing capture probe solvent. The cell is lysed by a lysis inducer in the capture probe to disperse single cell components into the medium. The lysed single cell components are transported to a mass spectrometer, where the lysed single cell components entering the mass spectrometer are spatially and temporally separated from any dispersed components of another single cell from the sample entering the mass spectrometer. Mass spectrometry is conducted on the lysed single-cell components. A system for analyzing single cells by mass spectrometry is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/685,740, filed Nov. 15, 2019, which claimspriority to U.S. Provisional Patent Application No. 62/781,048 filed onDec. 18, 2018, entitled “Rapid Native Single Cell Mass Spectrometry”,the disclosures of which are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No.DE-AC05-00OR22725 awarded by the U.S. Department of Energy. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to mass spectrometry, and moreparticularly to single cell mass spectrometry.

BACKGROUND OF THE INVENTION

Cell-to-cell differences are present in any cell population and, untilrecently, understanding of cell mechanisms relied on measure of cellpopulations in bulk. It is now generally understood that ensembleaverages may not represent individual cell function, hence the need forsingle cell resolution in genomics, epigenomics, transcriptomics,proteomics and metabolomics. Recent developments in single cell DNA andRNA sequencing have enabled the remarkable ability to monitor cellularlineage and cellular heterogeneity with single cell resolution. However,even isogeneic cells, having an identical genotype, can exhibitstochastic cellular heterogeneity. Non-genetic cellular differences inthe concentrations of metabolites and other components influence celldeath susceptibility, kinetics and even the mode of death in response touniform lethal drug exposure. In cancer treatment sequential rounds ofcytotoxic chemotherapies often kill a constant fraction of cells in atumor rather than an absolute number, suggesting either pre-existingcellular heterogeneity inhibiting treatment or the selective response ofa cellular sub-population to the treatment. Regardless, non-geneticcellular heterogeneity has clear ramifications on treatment and overallefficacy of therapeutics.

Detection of metabolites with single cell resolution is a significantanalytical challenge. Within a cell there are thousands of uniquechemical species present at low concentrations that can change rapidly.Unlike with single cell genomics and transcriptomics, there are noamplification strategies available for metabolites, thus highsensitivity is required. Also critical is high single cell samplingthroughput, which is needed to obtain enough single cell data tostatistically distinguish differences between cellular populations orsubpopulations. Flow cytometry, one of the most commonly used singlecell measurement techniques, uses fluorescence-based molecular probes toanalyze specific metabolites with extremely high throughput on the orderof thousands of cells/s but, due to overlapping spectroscopicsignatures, flow cytometry can only measure around a dozen moleculessimultaneously. Mass cytometry can target up to 48 different molecularcomponents using a combination of heavy-metal isotope-based molecularprobes, but this still represents only a small fraction of themetabolites present in a single cell. Unfortunately, untargetedmolecular analysis by flow and mass cytometry is not feasible becausethe researcher must know and select what molecules to investigate apriori.

Mass spectrometry (MS) is one of the most amenable technologies foruntargeted single cell analysis due to its high sensitivity, chemicalspecificity and speed. Several vacuum and probe-based MS techniques havebeen developed to enable comprehensive, untargeted chemical analysis ofsingle cells. Vacuum-based techniques include secondary ion MS (SIMS),matrix assisted laser/desorption ionization (MALDI), andlaser/desorption ionization (LDI) techniques, which, while extremelysensitive, require significant modification of the cell outside of itsnatural state, such as fixation and dehydration, to facilitate analysisunder vacuum. Several approaches, including live single cell MS, thesingle-probe MS and others, use manually guided probes such as pipettes,optical fibers and capillaries to measure cellular chemistry in situ andsometimes in vivo by ambient MS. However, low sampling throughput haslimited the value of most of these methods as the measure of each cellcan take an hour or more and requires significant user skill. A subsetof ambient MS techniques use laser pulses to dissect single cells. Thesetechniques are particularly useful for sampling cells in tissue.However, few of these techniques can measure cells from cell suspensionand typically have low sampling throughput and sensitivity.

MS-based techniques could, with further development, form the basis ofthe technology required for rapid, quantitative and untargeted singlecell chemical analysis. However, routine, quantitative measure of singlecell chemistry is still challenging. Moreover, current MS platformsrequire extensive sample preparation procedures or otherwise perturbcells from their native state in order to facilitate analysis, which canhave unforeseen ramifications on cellular chemistry. Chemical analysisof individual cells is a significant analytical challenge. Molecularconcentrations are low and high sampling throughput is needed to obtainenough single cell data to statistically distinguish differences betweencell populations or subpopulations. Single cell DNA and RNA sequencingusing next generation sequencing technology can take 2-3 days andthousands of dollars for each experiment, though recent advances arebeginning to make these techniques faster. Flow cytometry has beenheavily used for high throughput single cell analysis over the lastseveral decades. Unfortunately, molecular information obtained by flowcytometry is limited by the use of fluorescence-based molecular probesthat can target only one or a few molecular features simultaneously inan experiment due to overlapping spectroscopic signatures. Masscytometry (a combination of flow cytometry and inductively coupledplasma-mass spectrometry (ICP-MS)) has enabled up to 40 differentcomponents to be measured simultaneously in a single cell by usingheavy-metal isotope-based molecular probes. Fluorescence-based flowcytometry and mass cytometry techniques are among the highest throughputtechnologies for measuring single cell chemistry and are capable ofmeasuring thousands and hundreds of cells/s, respectively. However,untargeted molecular analysis is not feasible, i.e., the researcher mustknow and select what molecules to investigate a priori. Since cellularchemistry is complex, reactions can often proceed in an unanticipatedmanner and, thus, untargeted chemical analysis can provide criticalinsights. The addition of molecular probes also necessarily influenceschemical makeup of the cell, which may have unknown ramifications oncellular chemistry.

The high sensitivity, chemical specificity and speed of massspectrometry (MS) make it one of the most amenable technologies to usefor single cell analysis. Several MS-based techniques have beendeveloped to enable more comprehensive and untargeted chemical analysisof single cells, including the aforementioned mass cytometry technique.Nanomanipulation-based techniques are among the most sensitive and canmeasure cellular chemistry in situ. In these approaches cell contentsare manually extracted via a nanopipette and directly analyzed by MS;however, low sampling throughput has limited the value of these methodsas the measure of each cell can take the better part of an hour or moreand requires significant user skill. Using laser ablation (LA)-MSsystems, metabolic analysis can be achieved in a more rapid fashion, butof these techniques most have only measured major chemical constituentsfrom large 70×400 μm single cells that do not represent the size of themajority of mammalian cells. Liquid vortex capture (LVC)-MS can measurecells with a throughput of ˜15 s/cell, but the technique requires cellsto be dried onto a microscope slide, a process which may distort thechemistry of the cell in unknown ways. Another LA-MS technique, matrixassisted laser desorption/ionization-MS (MALDI-MS), combined withmicrofluidic chips that gently trap single cells on a surface has highthroughput (1-5 cells/s) and sensitivity for single cell analyses,however, intense matrix signals in the ≤500 mass per charge (m/z) rangemay interfere with small metabolites and pharmacological compounds ofinterest. Additionally, the application of matrix and other samplepreparation processes are time-intensive, costly, and may alter cellularchemistry from its native state.

Currently available mass spectrometric analysis techniques are not yetcapable of routinely and rapidly acquiring quantitative molecularinformation at the cellular level for a wide range of compound types(from small drugs and metabolites to large biopolymers like proteins).Moreover, many MS platforms require extensive sample preparationprocedures or otherwise perturb cells from their native state in orderto facilitate analysis, which can have unforeseen ramifications oncellular chemistry.

SUMMARY OF THE INVENTION

A method for analyzing single cells by mass spectrometry from a samplecontaining a plurality of cells includes the steps of providing aplurality of cells and a liquid medium and placing the cells and liquidmedium in a single cell isolation and ejection system. The liquid mediumcontaining a single cell is released from the single cell isolation andejection system. The liquid medium and single cell are captured in acapture probe containing a flowing capture probe solvent. The cell islysed with a lysis inducer in the capture probe to disperse single cellcomponents into the medium. The lysed single cell components aretransported to a mass spectrometer, where the lysed single cellcomponents entering the mass spectrometer are spatially and temporallyseparated from any dispersed components of another single cell from thesample entering the mass spectrometer. Mass spectrometry can then beconducted on the lysed single cell components.

The lysis can be induced by a capture probe solvent providing a lowerpartial pressure than the internal pressure of the cell to cause lysisof the cell. The capture probe solvent can include at least one selectedfrom the group consisting of methanol, ethanol, isopropranol, hexane,chloroform, dichloromethane, acetonitrile, and water.

The lysis can be induced by a chemical lysing agent. The chemical lysingagent can include at least one selected from the group consisting ofsodium dodecyl sulfate (SDS), Triton-X, NP-40 lysis buffer, RIPA lysisbuffer, Tween lysis buffer, CHAPS lysis buffer, and perfluorooctanoicacid.

The lysis can be induced by a liquid vortex formed by the flowingcapture probe solvent. The liquid vortex fluid rate can be 50-300 μL/minand can have a 0.5-20 second elution time. The liquid vortex can beformed by a coaxial probe.

The lysis can be induced by a voltage applied across the cell in thecapture probe solvent. The lysis can be induced by the application of0.2-1.5 volts across the cell for 1-10,000 μs to the cell in the captureprobe solvent.

The lysis can be induced by an acoustic wave emitted at the cell in thecapture probe solvent. The lysis can be induced by an acoustic waveemitted at the cell in the capture probe having a frequency of 15-20 kHzfor 0.1-10 seconds.

The release rate of the single cell isolation and ejection system can befrom 0.1 cell/s to 100 cells/s. The release rate of the single cellisolation and ejection system can be drop on demand. The liquid mediumcan include a cell culture medium.

A system for analyzing single cells by mass spectrometry can include asingle cell isolation and ejection system including a storage containerfor sample including a plurality of cells in a liquid medium. A captureprobe can include at least one solvent supply conduit for flowing acapture probe solvent, a capture probe exhaust conduit for receivingsingle cells released by the single cell isolation and ejection systemand conducting lysed cell components and capture probe solvent to a massspectrometer, and a lysis inducer. The system can further include a massspectrometer having an inlet connected to the capture probe exhaustconduit.

The lysis inducer can include a capture probe solvent. The capture probesolvent provides a lower partial pressure than the internal pressure ofthe cell to cause disruption of the cell. The lysis inducer can includea liquid vortex formed by the flowing capture probe solvent through thecapture probe. A co-axial probe can include a liquid supply and anexhaust conduit for forming a liquid vortex by the flowing capture probesolvent. The lysis inducer can include electrodes arranged on two sidesof the sampling capillary for applying a high voltage to cells in thecapture probe. The lysis inducer can include a chemical lysing agentinjector. The lysis inducer can include an acoustic wave generatorlocated around the outside of the sampling capillary for applying anacoustic wave to cells in the capture probe.

BRIEF DESCRIPTION OF THE DRAWINGS

There are shown in the drawings embodiments that are presently preferredit being understood that the invention is not limited to thearrangements and instrumentalities shown, wherein:

FIG. 1A is a schematic cross section of a system for analyzing singlecells by mass spectrometry; FIG. 1B is a magnified schematic crosssection of a capture probe and single cell isolation and ejectionsystem.

FIG. 2 is a magnified schematic cross section of a capture probe with aliquid vortex lysis inducer, and single cell isolation and ejectionsystem.

FIG. 3 is a magnified schematic cross section of a capture probe with anapplied voltage lysis inducer, and single cell isolation and ejectionsystem.

FIG. 4A is a magnified schematic cross section of a capture probe withan acoustic energy lysis inducer, and single cell isolation and ejectionsystem;

FIG. 4B is a magnified schematic depiction of area FIG. 4B in FIG. 4A.

FIG. 5 is a magnified schematic cross section of a capture probe with alysing agent injector, lysis inducer, and single cell isolation andejection system.

FIG. 6 is a plot of caffeine mass spectrometry signal intensity vs. Time(min) for caffeine droplets with a 90/10/0.1 (v/v/v)methanol/chloroform/formic acid solvent composition.

FIG. 7 is a plot of total mass spectrometry ion current (cps) vs. time(min) for Euglena gracilis cells.

FIG. 8 is a plot of total mass spectrometry ion current (cps) vs. time(min) for HeLa cells.

FIG. 9 is a zoomed plot of total mass spectrometry ion current (cps) vs.time (min) for Euglena gracilis cells.

FIG. 10 is a zoomed plot of total mass spectrometry ion current (cps)vs. time (min) for HeLa cells.

FIG. 11 is an image of a Euglena gracilis cell, before ejection.

FIG. 12 is an image of a HeLa cell, before ejection.

FIG. 13 is a mass spectral plot of intensity (cps) vs. mass-to-chargeratios (m/z) for a single Euglena gracilis cell.

FIG. 14 is a mass spectral plot of intensity (cps) vs. m/z for a singleHeLa cell.

FIG. 15 is a plot of intensity vs. time (min) for m/z 184, a PC Fragmention, Euglena Gracilis, and m/z 236, DGTS Fragment ion, ChlamydomonasReinhardtii.

FIG. 16 is an image of a collection of intact cells on glass afterspraying a cellular suspension in HSM media, without lysis.

FIG. 17 is an image of a collection of nebulized droplets with lysisfrom exposure to liquid vortex in the capture probe.

FIG. 18 is a plot of total ion chromatogram (TIC) peak area for CC503and CC125 cell type.

FIG. 19 is an extracted ion chromatogram (XIC) plot of relativeintensity vs. time (s) for m/z 732.6 corresponding to DGTS (34:4) andm/z 712.6, corresponding to the DGTS (32:0) internal standard.

FIG. 20 is a plot of peak area (a.u.) for nitrogen limited (−N) andnormal (+N) growth, for DGTS (34:4), measured from single cells and fromextracts.

FIG. 21 is a plot of peak area (a.u.) for nitrogen limited (−N) andnormal (+N) growth, for DGTS (34:3), measured from with single cell andextract.

FIG. 22 is a plot of peak area (a.u.) for nitrogen limited (−N) andnormal (+N) growth, for DGTS (38:4), measured from with single cell andextract.

DETAILED DESCRIPTION OF THE INVENTION

A method for analyzing single cells by mass spectrometry from a samplecontaining a plurality of cells includes the steps of providing aplurality of cells in a liquid medium and placing the cells and liquidmedium in a single cell isolation and ejection system. Liquid mediumcontaining a single cell is released from the single cell isolation andejection system. The liquid medium and single cell are then captured ina capture probe containing a flowing capture probe solvent. The cell islysed in the capture probe to disperse single cell components into themedium. The lysed single cell components are transported to a massspectrometer, where the lysed single cell components entering the massspectrometer are spatially and temporally separated from any lysedsingle cell of another cell from the sample entering the massspectrometer. Mass spectrometry is conducted on the lysed single cellcomponents. The lysis can be induced in the capture probe by differentmethods and structures.

A system for analyzing single cells by mass spectrometry can include asingle cell isolation and ejection system including a storage containerfor a sample including a plurality of cells in a liquid medium. Acapture probe includes at least one solvent supply conduit for flowing acapture probe solvent, a capture probe exhaust conduit for receivingsingle cells released by the single cell isolation and ejection systemand conducting lysed cell components and capture probe solvent to a massspectrometer, and a lysis inducer. The system can further include a massspectrometer having an inlet connected to the capture probe exhaustconduit. The lysis inducer can vary. More than one type of lysis inducercan be incorporated into the capture probe.

The lysis inducer can include an appropriate capture probe solvent. Thecapture probe solvent provides a lower partial pressure than theinternal pressure of the cell. The lysis is induced by the capture probesolvent providing a lower partial pressure than the internal pressure ofthe cell to cause disruption and lysis of the cell. The capture probesolvent can be any suitable solvent. The capture probe solvent caninclude at least one selected from the group consisting of methanol,ethanol, isopropanol, hexane, chloroform, dichloromethane, acetonitrileand water.

The lysis inducer can include a chemical lysing agent. The chemicallysing agent can be any suitable lysing agent. The chemical lysing agentcan include at least one selected from the group consisting of sodiumdodecyl sulfate (SDS), Triton-X, NP-40 lysis buffer, RIPA lysis buffer,Tween lysis buffer, CHAPS lysis buffer, and perfluorooctanoic acid. Thechemical lysing agent can be dispersed in the capture probe solvent. Thelysis inducer can include a chemical lysing agent injector. The positionand construction of the chemical lysing agent injector can vary.

The lysis can be induced by a liquid vortex formed by the flowingcapture probe solvent. The lysis inducer can include structure forforming the liquid vortex from the flowing capture probe solvent throughthe capture probe. The lysing vortex can be created by any suitablestructure. The liquid vortex can be formed by a coaxial probe in whichcapture probe solvent flows from one coaxial solvent supply flow path,overflows a common open end, and enters the coaxial solvent exhaust flowpath in a vortex flow pattern. Other vortex inducing structure in theexhaust flow path is possible. The coaxial probe can include a liquidsupply and an exhaust conduit for forming a liquid vortex formed by theflowing capture probe solvent. The liquid vortex so created can havevarying characteristics, but in general should provide sufficientkinetic energy to lyse the cell. The liquid vortex can have a fluid flowrate of 50-300 μL/min. The liquid vortex can have a fluid flow rate of50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, 290, 295 and 300 μL/min, or within a range of anyhigh value and low value selected from these values. The liquid of thecapture probe solvent can have an elution time of 0.5-20 seconds. Theliquid of the capture probe solvent can have an elution time of 0.5, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, 20 seconds, or within a range of any highvalue and low value selected from these values.

The lysis inducer can include electrodes arranged on two sides of thesampling capillary for applying a high voltage to cells in the captureprobe. The lysis is induced by the application of the voltage to thecapture probe solvent carrying the cell. The construction of theelectrodes can vary, and the voltage applied can vary. The voltage canbe 0.2-1.5 volts across the cell. The voltage can be 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 volts, or within arange of any high value and low value selected from these values. Thetime that the voltage is applied can vary. The voltage can be applied tothe cell in the capture probe solvent for 1-10,000 μs. The voltage canbe applied to the cell in the capture probe solvent for 1, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 μs, orwithin a range of any high value or low value selected from thesevalues.

The lysis inducer can include an acoustic wave generator located aroundthe outside of the sampling capillary for applying an acoustic wave tocells in the capture probe. The lysis is induced by the acoustic waveemitted at the cell in the capture probe. The construction of theacoustic wave generator can vary. The frequency of the acoustic wave canvary. The acoustic wave can have a frequency that is near the resonantfrequency of the cell membrane. The acoustic wave can have a frequencyof 15-20 kHz. The acoustic wave can have a frequency of 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5 or 20 kHz, or within a range of anyhigh value and low value selected from these values. The amount of timethat the acoustic wave is applied to the cell can vary. The acousticenergy can be applied to the cell for 0.1-10 seconds. The acousticenergy can be applied to the cell for 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 seconds, or withina range of any high value and low value selected from these values.

The release rate of the cells from the single cell isolation andejection system can vary. The release rate of the single cell isolationand ejection system can be from 0.1 cell/s to 100 cells/s. The releaserate of the single cell isolation and ejection system can be 0.1, 1, 10,20 30, 40, 50, 60, 70, 80, 90, and 100 cell/s, or within a range of anyhigh value and low value selected from these values. The release rate ofthe single cell isolation and ejection system is drop on demand. Theliquid medium comprises a cell culture medium. The design andconstruction of the single cell isolation and ejection system can vary.

Droplet ejection using an inkjet printer-like mechanism as the singlecell isolation and ejection device is an efficient means for isolationof single cells from bulk in a label free and non-contact manner and canutilize a microfluidic dispenser chip and a piezoelectric actuator thatcan be activated on demand. Once activated, the piezoelectric actuatorcompresses the dispenser chip causing release of a single droplet. Thesize of the ejected droplet (tuned to be slightly larger than a typicalcell) and use of video microscopy to visualize cells in the microfluidicdispenser chip before they are ejected ensure that each droplet containsone cell. Cells remain viable after the droplet ejection process andthese techniques are often used to populate new cell cultures stemmingfrom a single genetic variant. The invention uses the single cellisolation and ejection technology, such as a single cell printer (SCP),to isolate and transfer single cells for analysis by mass spectrometry.

Liquid vortex capture (LVC) mass spectrometry or LVC-MS is acontinuously flowing liquid solvent extraction, atmospheric pressure MStechnology. The LVC-MS technique uses a co-axial tube design having astable liquid vortex drain maintained at the sampling end of the probewhen operated under optimized solvent and nebulizer gas conditions.Sample that comes in contact with the liquid vortex surface is captured,analyte molecules are extracted from the sample and transported to theionization source of the mass spectrometer over a few seconds.Subsequently, the analyte is ionized using electrospray ionization(ESI), atmospheric pressure chemical ionization (APCI), or nearly anyliquid-based ionization source and is detected by MS.

The single cell isolation and ejection system and capture probe lysisand MS technologies are coupled together by ejection of single cellsdirectly into the lysis inducing capture probe. The droplet containing asingle cell ejected by the single cell isolation and ejection systemfalls towards the capture probe, located directly below the site ofdroplet ejection. The droplet is captured by the flowing solvent of thecapture probe where the cell is lysed by the lysis inducer. For example,upon being exposed to solvent (e.g., methanol, chloroform, etc.), thecell wall can rupture due to the change in osmotic pressure. Thecontents of the cell can be further extracted by the solvent while intransport to the ionization source of the mass spectrometer (˜6 s). Fromthere, the analyte molecules are ionized and then measured using MS inan untargeted (e.g., time-of-flight (TOF)-MS) or targeted (e.g., tandemMS) manner. The technique described in the invention is able toefficiently extract cellular contents while minimizing sample dilution,enabling the sensitivity needed to measure lipids and metabolites insingle cells. Since the capture probe is continuously operating withflowing solvent, the probe is self-cleaning, and signal carryover fromone cell to another is removed and background signal from solvent ionsor ions due to media can be easily determined. Cells remain in their‘natural’ state (i.e., in this case in growth media) until the point oflysis and chemical analysis by MS. Further, the technology requires nomolecular labeling or other sample preparation protocols. The massspectrometer and ionization source operate in the exact same manner asthat of direct infusion-ESI experiments often used for measuring cellchemistry in aggregate.

FIG. 1A is a cross section of a system 10 for analyzing single cells bymass spectrometry. The system 10 includes a capture probe 14 and asingle cell isolation and ejection system 18. The capture probe 14 canhave differing dimensions. In the embodiment shown particularly in FIG.1B, the capture probe 14 includes an outer tubular housing 20 having anopen interior 24. The capture probe housing 20 includes an open end 28communicating with the open interior 24. Within the open interior 24 isan exhaust conduit 32 having an open end 34 and an open interior 36. Thedimensions and location of the exhaust conduit 32 can vary. In theembodiment shown, the exhaust conduit 32 is placed in substantiallycoaxial relationship with the outer housing 20. The open end 34 can bepositioned within the open interior 24 in various locations, but ingeneral will be offset from the open end 28 of the outer housing 20.This ensures that fluid flowing through the open interior 24 will bedrawn into the open end 34 of the exhaust conduit 32.

The single cell isolation and ejection system 18 can have differingdesigns. Such devices are also known as single cell printers. In theembodiment shown, a housing 38 includes a container 39 for receiving aplurality of cells in a liquid medium. An isolation housing 40 includesan isolation chamber 44 that receives cells 64 and liquid medium fromthe container 39 (FIG. 2 ). An ejection opening 48 is dimensioned topass single cells 64 in droplets of liquid medium 70.

Connections are provided to deliver solvent to the capture probe 14 andto remove solvent containing sample from the capture probe 14 andtransport it to a mass spectrometer. The exhaust conduit 32 cancommunicate with mass spectrometer inlet conduit 52, attachment 56 andmass spectrometer port 60. A solvent inlet conduit 66 supplies solventto the capture probe 14 and to the interior channel 24 of the captureprobe housing 20. A manifold fitting 68 can be provided to convenientlyprovide connections and passage for the exhaust conduit 32, massspectrometer inlet conduit 52, solvent inlet conduit 66, and captureprobe housing 20. Other designs are possible.

The capture probe 14 includes a lysis inducer. The lysis inducer breaksup at least the cell wall or membrane of the single cell 64 whenreceived from the single cell isolation and ejection system 18. Thesingle cell 64 is received through the open end 28 of the capture probe14. Solvent flows through the open interior 24 of the capture probehousing 20 in the direction shown by arrow 74 (FIG. 2 ) toward the openend 28 of the capture probe 14. A suitable pump can be applied to theexhaust conduit 32 to draw solvent into the open interior 36 of theexhaust conduit 32. Solvent will flow in the direction of arrow 92 fromthe capture probe to the mass spectrometer. The solvent flow rate in theopen interior 24 can be balanced with the solvent flow rate in the openinterior 36 such that solvent does not overflow the open end 28 of thecapture probe housing 20. The single cell 64 enters the open end 28 ofthe capture probe 14 and is drawn into the open interior 36 of theexhaust conduit 32. The cell membrane of the single cell 64 is lysed bythe lysis inducer as the single cell 64 traverses the exhaust conduit32.

The manner in which the lysis inducer operates, and its position andconstruction, can vary. There is shown in FIG. 2 a solvent vortex 80that operates as a lysis inducer. The solvent vortex 80 is created bythe design and construction of the outer housing 20, exhaust conduit 32,and the relative flow rates in the open interior 24 and the openinterior 36. The single cells 64 in droplets 70 of liquid medium entersthe open end 34 of the exhaust conduit 32 and contacts the vortex 80.The kinetic energy of the vortex 80 breaks apart the single cell 64, atleast the cell membrane. Cell pieces 84 are produced. It is alsopossible that the kinetic energy of the vortex 80 further breaks apartthe cell pieces 84 into smaller cell fragments 88.

FIG. 3 is a magnified cross section of a capture probe with an appliedvoltage lysis inducer. The applied voltage license inducer 100 includeselectrodes 101 and 102 which are positioned so as to induce a voltagepotential across the solvent within the open interior 36 of the exhaustconduit 32. The electrodes 101 and 102 are shown within the captureprobe 14 however, it is also possible to position the electrodeselsewhere in the capture probe 14 as within the outer housing 20 oroutside the outer housing 20. The applied voltage will act on singlecells 64 traversing the exhaust conduit 32 to break the cell membraneand create cell pieces 106, and possibly also cell fragments 110.

It is also possible to use acoustic energy as a lysis inducer. There isshown in FIG. 4A is a magnified cross section of a capture probe with anacoustic energy lysis inducer 120 that is comprised of an acousticenergy transducer 122 and possibly an additional acoustic energytransducer 124. Acoustic energy transducers are well-known and differentsizes and shapes and locations are possible. As shown particularly inthe expanded view of FIG. 4B, an acoustic wave 128 can be generated bytransducer 122 and an acoustic wave 130 can be generated by transducer124. The acoustic energy is transferred through the exhaust conduit 32and in parts the acoustic wave to the solvent flowing through theexhaust conduit 32. The acoustic wave energy results in the single cell64 being lysed into cell pieces 132 and possibly also cell fragments 136as the single cell 64 traverses the exhaust conduit 32 and the acousticwave energy.

The lysis inducer can also comprise a lysing agent. Lysing agents arechemical compositions which react with components of the cell wall ormembrane to disrupt the cell membrane and release cellular components.The lysing agent may further act to degrade the cellular components intosmaller cellular pieces and fragments. The lysing agent can be selectedfor this purpose. FIG. 5 is a magnified cross section of a capture probe14 with a lysing agent injector. The precise construction and locationof the lysing agent injector can vary, and different embodiments areshown in FIG. 5 . A lysis agent injector 150 can receive lysing agentfrom source 154 and releases the lysing agent at opening 158 into theexhaust conduit 32. The lysing agent will contact the single cell 64within the exhaust conduit 32 to lyse the cell 64 into cell pieces 204and possibly cell fragments 210. Other lysing agent injectors arepossible. A lysing agent injector 164 can receive lysing agent fromsource 168 and releases the lysing agent into the open interior 24 ofhousing 20 at an opening 172. It is also possible to inject the lysingagent into the solvent prior to the solvent reaching the capture probe14. There is shown in FIG. 5 another alternative embodiment in which thesolvent supply line 180 receives solvent flow stream 184. A lysing agentinjector 190 receives lysing agent flow 194 and injects the lysing agentinto the solvent flow stream 184. The combination of solvent and lysingagent is then injected at opening 200 into the open interior 24 of thehousing 20, such that the lysing agent will contact cells 64.

The invention was tested using a single cell isolation and ejectionsystem which incorporated small droplet piezoelectric ejection followedby capture of the droplet into a liquid vortex capture probe, celllysis, and transport to the mass spectrometer. Once exposed to anappropriate solvent the cell is lysed, extracted and analyzed by MS. Themethod was validated by measuring the lipid composition of microalgae,Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLacells in their native growth media. ChRe and EuGr microalgae mixedtogether in the same solution were able to be differentiatedcell-by-cell based on measured lipid profiles. Numerousdiacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC),monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol(DGDG) lipids were observed in single cells. Continuous solvent flowensures that cells are analyzed rapidly and no signal carryover betweencells is observed. ChRe and EuGr microalgae mixed together in the samesolution were differentiated cell-by-cell in real-time based ondifferences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) andphosphatidylcholine (PC) lipids measured in each cell. Several DGTSlipids present in ChRe were quantified with single cell resolution bynormalizing lipid mass spectrometric signal to a DGTS(32:0) internalstandard added to the LVC probe solvent during analysis. Quantitativepeak areas were validated by comparing to bulk lipid extracts. Peak areadistributions comprised of hundreds of cells were compared for ChReafter 5 days of nitrogen-limited and normal growth conditions, whichshow clear differences and the ability to resolve cellular populationdifferences with single cell resolution. A total of >20,000 cells wereanalyzed in these experiments.

LC-MS CHROMASOLV® methanol+0.1% formic acid (FA), chloroform, and waterwere purchased from Sigma-Aldrich (St. Louis, MO, USA). EuGr cells werepurchased from Carolina Biological (Burlington, NC, USA). ChRe CC-503cw92 mt+ and CC-125 wild type mt+[137c] were purchased from theChlamydomonas Resource Center (St. Paul, MN, USA). Algae was grown inhigh salt medium (HSM) media under constant shaking, light, andtemperature (25° C.). In normal growth (+N) and nitrogen deprived (−N)ChRe growth experiments, cells were grown for 5 days under constantshaking, light, and temperature (25° C.) in HSM and HSM made withoutammonium chloride, respectively. Cell counts were measured using ahemacytometer. HeLa cells were purchased from ATCC (Manassas, VA, USA).DGTS(32:0) was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

The system was comprised of a commercially available single cellisolation and ejection device, the Single-Cell Printer™ (Cytena Gmbh,Freiburg, Germany) coupled with a modified sampling capture probe. Thesingle cell isolation and ejection device is a benchtop sized, automateddrop-on-demand cell printer utilizing the inkjet-like dispensingprinciple combined with optical microscopy to ensure onecell-per-droplet is ejected. Using this device>95% of ejected dropletscontain a single cell. In practice, shadows along the wall of themicrofluidic sample cartridge can obfuscate a cell from an imagerecognition algorithm potentially resulting in two cells in a droplet,thus, single cell dispensing efficiency is <100%. The instrument used inthis study is capable of ejection of droplets at a controllablethroughput of up to ˜30 droplet/s and can isolate cells ranging in sizebetween 5-30 μm diameter.

The single cell isolation and ejection system dispenser and imagerecognition settings need to be adjusted for each sample cartridge usedand for the cells being investigated. For each experiment, a disposablemicrofluidic sample cartridge was filled with 50 μL of cell solution andwas mounted onto the dispenser. The dispenser's piezo-electric plungergenerates ˜70 μm droplets in diameter from the cartridge without beingin direct contact to the sample. A video monitor and image recognitionalgorithm detected cells in the droplet ejection area of the samplecartridge. Droplets not containing a single cell, or containing two ormore cells, are removed using a vacuum shutter system located directlyunderneath the nozzle, while those droplets that do contain a singlecell are allowed to pass. Once the sample cartridge is attached to thedispenser, the cell image recognition target area is set to the regionsurrounding the orifice of the sample cartridge. The image algorithm wastuned to accurately identify cells in the sample cartridge. A 5-25 μmcell diameter, 0.4-1.0 roundness and 30 greyscale thresholds were usedfor all experiments. The piezoelectric instroke depth (8-12 μm) andspeed (90-110 μm/ms) were tuned to eject droplets reproducibly,evidenced by imaging of the droplets after ejection.

The capture probe consisted of a 1.12 mm i.d. 1.62 mm o.d. stainlesssteel outer annulus connected to a 5-port PEEK manifold (IDEX Health andScience). The inner annulus was a 20-cm long, 0.178 mm i.d., 0.794 mmo.d. PEEK capillary (IDEX Health and Science) that directly connected tothe ion source of the mass spectrometer.

The single cell isolation and ejection system was placed on top of aSciex TripleTOF® 5600+ mass spectrometer and the capture probe wasattached onto the base of the single cell isolation and ejection system.The sample cartridge was aligned 1 mm directly above the capture probe,ensuring that all droplets ejected for analysis are captured by theprobe. The capture probe solvent flowrate varied with solventcomposition. Mass spectrometer scan settings were optimized for eachexperiment.

Single cell mass spectrometry data was analyzed using Matlab 2018b(Mathworks, Natick, MA, USA). SCPAssistant v1.20© software was used forreal-time cell differentiation and was written in Delphi 3. All massspectra were background subtracted from spectra acquired from dropletscontaining just cellular media.

The event that a single droplet contains a cell is a Poisson pointprocess such that the cell concentration and droplet ejection frequency(i.e., flow rate) are necessary to predicting the throughput of singlecell droplet ejection by the SCP. A droplet ejection speed of 30droplets/s was used which corresponds to a ˜0.32 μL/min flow rate. Cellconcentrations around 1·10⁶ cells/mL generally yielded a high enough SCPspeed to reach the throughput limitation of mass spectrometric analysis.

A 2.5 μM solution of caffeine dissolved in water was used to validatecapture probe-droplet alignment and evaluate the analytical metrics ofthe system such as sensitivity, reproducibility, peak width, peak shape,and sampling throughput. The caffeine droplet experiments used a 150μL/min flow rate with a 90/10/0.1 (v/v/v) methanol/chloroform/formicacid solvent composition. The capture of 50 consecutive dropletscorresponding to 163 amol of caffeine/droplet spaced 1 s apart measuredusing single reaction monitoring (SRM) ESI-MS of caffeine (m/z 195.1[M+H]⁺→138.1 [M-C₂NOH₃+H]⁺), 0.10 s accumulation time, 5500 Velectrospray voltage (IS), 70 V declustering potential (DP), 27 eVcollision energy (CE), 400° C. turbo heater temperature (TEM), andGS1=90 and GS2=60 N₂ gas settings. These tests resulted in reproduciblepeaks (RSD=5.7%)˜1.2 s wide (baseline-to-baseline, see FIG. 6 ).Continuous solvent flow ensures that there is no signal carryoverbetween droplets, evidenced by a return to baseline solvent ion signalsbetween droplets. Based on these metrics the maximum sampling throughputof this single cell printer/liquid vortex capture/mass spectrometry(SCP-LVC-MS) system would be ˜1 cell/s.

To evaluate the ability of the system to measure single cell chemicalprofiles CC-125 wild type mt+[137c] ChRe microalgae which are ˜5-15 μmin diameter were used. The ChRe cell suspension was pipetted, withoutdilution or other alteration to the cell suspension, into the singlecell isolation and ejection system sample cartridge, placed on thedispenser and settings tuned for droplet ejection. Only dropletscontaining a single cell were allowed to fall into the capture probe.Images of the sample cartridge immediately before and after dropletejection of a cell were taken and used to verify proper ejection. Thesignal peak width due to each cell was measured to be ˜1.2 s, identicalto droplets of caffeine measured previously and shown in FIG. 6 .Multiple MS scans can be acquired within this duration. Dozens ofdiscrete ions were observed in the TOF mass spectrum. Ions werepredominantly diacylglyceryltrimethylhomo-Ser (DGTS) andmonogalactosyldiacylglycerol (MGDG) lipids, based on separate tandem MSexperiments and SWATH-MS scans which show an abundance of the DGTSheadgroup and MGDG fragment ions. Specific lipids identified includeDGTS(34:4) (m/z 732.6 [M+H]⁺), DGTS(34:3) (m/z 734.6 [M+H]⁺), DGTS(38:4)(m/z 783.5 [M+H]⁺), MGDG(34:7) (m/z 767.5 [M+Na]⁺) among other smallmolecules including pheophytin a (m/z 871.7 [M+H]⁺), chlorophyll a (m/z893.7 [M+H]⁺) and chlorophyll b (m/z 906.7 [M+H]⁺).

Separate experiments were conducted to validate that additional celltypes, including mammalian cells, can be measured with the SCP-LVC-MStechnique. Single cell mass spectra were acquired for an additionalmicroalgae, EuGr, as well as for HeLa cells, the latter a commonly usedhuman epithelial cell line derived from cervical cancer. Cellsuspensions of each cell type were pipetted directly from their cultureor storage medium (HSM and phosphate buffered saline (PBS) formicroalgae and HeLa cells, respectively) into respective SCP samplecartridges and sampled by the LVC probe. Other than dilution of the HeLacell suspension no sample preparation such as filtering, centrifugation,fixing was used in either experiment prior to analysis.

Single cell analysis of ChRe used a 150 μL/min flowrate with a 90/10/0.1(v/v/v) methanol/chloroform/formic acid solvent composition. The massspectrometer was configured to acquire TOF mass spectra (m/z 700-1000,0.05 s accumulation time, IS=5500 V, DP=70 V, TEM=400° C., GS1=90 andGS2=60) as well as three SWATH mass spectra. The three SWATH-MSacquisition scan windows used in this experiment were SWATH spectra#1=m/z 700-800, SWATH spectra #2=m/z 800-900, and SWATH spectra #3=m/z900-1000. A m/z 100-1000 scan range, 0.03 s accumulation time, IS=5500V, DP=35 V, CE=48 eV, collision energy spread (CES)=15 eV, TEM=400° C.,GS1=90 and GΩ=60 nebulizer gas settings were used for each SWATHexperiment. The TOF and three SWATH scans were acquired continuouslyevery 0.2 s.

Single cell analysis of EuGr and HeLa cells used a 150 μL/min flowratewith a 90/10/0.1 (v/v/v) methanol/chloroform/formic acid solventcomposition. The mass spectrometer was configured to acquire TOF massspectra (m/z 700-1000, 0.10 s accumulation time, IS=5500 V, DP=70 V,TEM=400° C., GS1=90 and GS2=60). The total ion chromatogram (TIC) ofEuGr is shown in FIG. 7 . The TIC of HeLa is shown in FIG. 8 . FIG. 9and FIG. 10 show zoomed views of the TIC traces for EuGr and HeLaexperiments, respectively. Images of (d) EuGr and (e) HeLa cells beforeejection are shown in FIG. 11 and FIG. 12 , respectively, beforeejection. EuGr cells are ˜20 μm in diameter, are elliptically shaped andare highly mobile as evidenced by their movement in the sample cartridgeduring SCP-LVC-MS analysis. The activity of the cells highlight thatthey are unperturbed from their natural state up until the point of LVCprobe capture.

Mass spectra of the EuGr are shown in FIG. 13 , and for the HeLa cell inFIG. 14 . TOF-MS spectra from EuGr cells (FIG. 13 ) contain ions similarto ChRe (such as pheophytin a, chlorophyll a and chlorophyll b). Mostpredominantly, MGDG(34:7) (m/z 762.7 [M+NH₄]⁺), phosphatidylcholine(PC)(40:8) (m/z 830.6 [M+H]⁺) and digalactosyldiacyl-glycerol(DGDG)(34:4) (m/z 930.6 [M+H]⁺) lipids were observed. The image and massspectrum of a single HeLa cell (˜15 μm diameter) are shown in FIG. 12and FIG. 14 , respectively. 500 single cell mass spectra were acquiredin one experiment. Identified ions include PC(34:1) (m/z 760.7 [M+H]⁺),PC(34:2) (m/z 780.6 [M+Na]⁺), PC(34:1) (m/z 782.6 [M+Na]⁺) and PC(34:0)(m/z 784.6 [M+Na]⁺) among many other ions that were unidentified.

Together this data highlights the capability to measure single cellchemistry from algae and mammalian cell types. No sample preparationmeasures were taken other than dilution, but, at most, cells may need tobe diluted/concentrated or media filtered for large particulates tofacilitate high sampling throughput and mitigate the potential forclogging of the SCP sample cartridge, respectively. The HSM and PBSmedia generally had a negligible effect on single cell mass spectra inthis m/z range (700-1000) and could be evaluated by sampling emptydroplets of each media. The small size of the droplet (70 μm) relativeto the probe (1 mm) minimizes the impact of high salt-induced ionsuppression effects, as has been seen in prior work. Measure of emptydroplets yielded little to no signal of the lipids measured in thecells, indicating that lipid signal is from a cell lysed in the LVCprobe rather than cellular exudates in solution.

Unique to the system is the ability to measure single cells in anuntargeted manner with high sampling throughput relative to otheruntargeted MS single cell approaches. The acquisition of 1000 ChRe cellswere taken with an overall sampling throughput of ˜2.5 s/cell. Theanalyte signal from each cell was fully resolved baseline-to-baselinewithout cross contamination between cells. The theoretical throughput ofthe system is ˜1 s/cell based on the ˜1.2 s signal peak width of asingle droplet containing caffeine. In practice, throughput is slowerbecause cells are not aligned optimally in solution, as not everydroplet contains a single cell and thus are removed by the vacuumshutter of the SCP.

To demonstrate that a single cell is being measured in each droplet,(1:1 v/v) cell suspensions of ChRe were mixed with EuGr and single cellmass spectra were acquired. These two cell types can be chemicallydifferentiated by the strong presence of DGTS lipids in ChRe and PClipids in EuGr. DGTS and PC lipid classes were targeted using tandem MSby monitoring for the lipid headgroup fragment ions m/z 236 and m/z 184indicative of DGTS and PC lipids, respectively. FIG. 15 shows extractedion chromatograms of fragment ions indicative of PC and DGTS lipidsfound in EuGr and ChRe cells, respectively. Cell types were clearlyseparated using DGTS and PC fragment ion signals. Cell identificationwas confirmed using a series of microscopy images of the dropletejection area immediately before ejection of the cell. Of the 500 cellsanalyzed in this preliminary experiment, comparison of DGTS and PCfragment ion peak areas were able to identify each cell type with 100%accuracy.

The single cell analysis of EuGr and ChRe cell mixture shown in FIG. 15used a 100 μL/min flowrate with a 90/10/0.1 (v/v/v)methanol/chloroform/formic acid solvent composition. The massspectrometer was configured to acquire SWATH mass spectra with a m/z700-800 acquisition scan window. A m/z 100-300 scan range, 0.05 saccumulation time, IS=5500 V, DP=35 V, CE=48 eV, CES=15 eV, TEM=400° C.,GS1=90 and GS2=60 nebulizer gas settings were used. Extracted ionchromatograms (XIC) of ions at m/z 184.15 (PC headgroup) and m/z 236.24(DGTS headgroup) were used.

To show that such chemical classifications could be conducted inreal-time a software solution, SCPAssistant v1.20© was used to extractsingle cell mass spectra while the experiment was in progress. As aproof-of-concept experiment, real-time cellular classification of theChRe and EuGr cell mixture was conducted for the acquisition of 87cells. Using lipid headgroup ions m/z 236 and m/z 184, automaticclassification of each cell was achieved within ˜3 s after acquisition.These identifications were confirmed through imaging of the samplecartridge immediately before and after droplet ejection and DGTS and PCfragment ions XICs. Identification was 100% accurate for this cellmixture. Such on-line chemical classification could conceivably be usedto classify and differentiate any cellular mixture (e.g., in blood orfor tumors) using a reference library of cell mass spectra.

Nutrient deprivation is a known route to perturb lipid and metaboliteconcentrations of ChRe. Quantitation requires the cell to be fullylysed, the molecular constituents fully extracted, and matrixsuppression effects, due to cellular media or from cellular metabolites,to be normalized. Collection of LVC-electrosprayed material after singlecell droplet ejection and exposure to LVC solvent indicate that cellsare efficiently lysed in the system. FIG. 16 shows cell lysis after LVCcapture and electrospray, and the collection of intact cells ontomicroscope slide after spraying cells in HSM medium directly (i.e. nosolvent or voltage). FIG. 17 shows the collection of nebulized dropletsafter SCP-LVC with solvent (methanol). No evidence of intact cells wasobserved, indicating cells are lysed after exposure to LVC probe solventand nebulization.

FIG. 18 shows average integrated TIC peak areas between CC503 (cellwall-less) and CC125 (normal) ChRe cells. Signal levels from CC125 werenot significantly lower than CC503 (wall-less mutant), indicating thatCC125 cells are lysed even with the presence of a cell wall. Tonormalize for ion suppression effects, 1 nM of DGTS(32:0) was added tothe LVC sample probe solvent and was monitored continuously. TheDGTS(32:0) lipids was selected because the ion was not observed insingle cell mass spectra and was the nearest standard to the DGTS lipidclass that was commercially available. CC125 measured peak area is notsignificantly lower than CC503 measured peak area with >95% confidence,indicating that the presence of a cell wall in CC125 does not appear tonegatively influence LVC-MS analysis. Physical examination of cells andthe overall agreement of average cellular intensities all confirm near100% lipid extraction of soluble analytes from single cells in theseexperiments.

FIG. 19 shows the XIC of m/z 732.6, corresponding to DGTS(34:4) fromsingle cells, and m/z 712.6, corresponding to the DGTS(32:0) internalstandard. Average quantitative peak areas are shown for DGTS(34:4) (FIG.20 ), DGTS(34:3) (FIG. 21 ) and DGTS(38:4) (FIG. 22 ) lipids derivedfrom bulk lipid extract (light grey) and single cell (dark grey) data ofcells undergoing nitrogen limited (−N) and normal (+N) growth. Anasterisk “*” indicates the two means are equivalent (p<0.05). The singlecell analysis of +N and −N ChRe cells shown in FIGS. 19-22 used a 150μL/min flowrate with a 90/10/0.1 (v/v/v) methanol/chloroform/formicacid+2 μL/min 1.4 μM DGTS(32:0) solvent composition. The massspectrometer was configured to acquire TOF mass spectra (m/z 700-1000,0.10 s accumulation time, IS=5500 V, DP=70 V, TEM=400° C., GS1=90 andGS2=60).

Average single cell measurements were compared to bulk lipid extractsusing the Bligth and Dyer extraction protocol, a common method tomeasure aggregate lipid composition. The modified Bligth and Dyerextraction protocol was as follows. 0.5 mL of nitrogen deprived (−N)cells, normal growth cells (+N), and HSM media (blank) were pipettedinto glass vials. 3 mL 2:1 (v/v) chloroform/methanol was added to eachvial. 15 μL of 21 μM DGTS(32:0) was added to each vial (internalstandard). Then each vial was vortexed for 30 s. 1 mL of chloroform wasthen added to each vial and vortexed for 30 s. 1 mL of water was addedto each vial and then was vortexed for 30 s. Each vial was centrifugedfor 5 min at 2260 RPM (Eppendorf 5430 centrifuge, Hamburg, Germany). Thelipid extract was pipetted out into an additional glass vial. Twoadditional extracts were performed by adding 1 mL of chloroform,vortexing for 30 s, centrifugation and removal of lipid extract. Thecollected lipid extract was dried using N₂ gas and then re-suspended in0.5 mL 90/10/0.1 (v/v/v) methanol/chloroform/formic acid. 2 μL/min ofextract mixed with 150 μL/min 90/10/0.1 (v/v/v)methanol/chloroform/formic acid were measured by ESI-MS. The massspectrometer was configured to acquire TOF mass spectra (m/z 700-1000,0.10 s accumulation time, IS=5500 V, DP=70 V, TEM=400° C., GS1=90 andGS2=60).

The dilution of the cell in the LVC probe solvent can be calculatedusing the LVC flowrate, generally 150 μL/min, and the single cell signalpeak width, ˜1.2 s, which indicates that each cell was contained within3 μL of solvent. Given the Gaussian-like peak shape of the signalprofile, majority of analyte (˜1 sigma/68%) lies within 0.4 s or ˜1 μLof solvent.

Quantified peak area/cell of DGTS(34:4), DGTS(34:3), and DGTS(38:4)lipids after 5 days of growth were calculated from the acquisition of556 and 704 single cells for +N and −N growth conditions, respectively(FIGS. 20-22 ). Lipid extract and average single cell data could beconsidered equivalent for all but the +N, DGTS(38:4) experiment. Forequivalence testing a standard error at 95% confidence of the extractmeasurement was used to define the confidence level limits for singlecell measurements to be considered equivalent. Absolute quantitationcould be achieved by using an external calibration curve of eachmolecule in question, but in the present case standards of thesespecific lipids were not available.

There were notable mass spectral differences between +N and −N growth.Cell suspensions were notably different in color with +N growth beingsignificantly greener. This is consistent with greater pheophytin a,chlorophyll a and chlorophyll b peak areas observed in +N cells. Cellgrowth differed greatly between nutrient conditions, with −N growth cellnumber concentrations remaining relatively stagnant over the 5 days ofthe experiment. Cell size distributions, measured using the imagerecognition algorithm of the Single-Cell Printer, were similar betweenboth growth conditions. The cell size distribution data is influenced bythe image recognition algorithm settings, which in this case were set toallow acquisition of cells with diameters between 5-25 μm. Single cellpeak area histograms from DGTS(34:4), DGTS(34:3), DGTS(38:4),MGDG(34:7), pheophytin a, and chlorophyll a ions observed in ChRe for +Nand −N experiments and these ions peak areas were significantly greaterfor normal growth conditions. DGTS(34:4) and DGTS(34:3) lipids had verydifferent distributions despite the similarity between molecules. Thiswas also apparent in cell extract data.

These data demonstrate that the invention enables untargeted, highthroughput and quantitative mass spectrometric analysis of single cellsin their native media. The invention demonstrates the untargeted andtargeted acquisition of single cell mass spectra of microalgae and HeLacells with high throughput (˜2.5 s/cell). Real-time chemicalclassification of single cells can be achieved, using software, based onchemical differences observed in mass spectra. The ability to acquireSWATH spectra in addition to TOF-MS spectra can help to chemicallydifferentiate more complex cell systems. The system was used toquantitate lipids and show their distribution in single cells grownunder +N and −N conditions through the incorporation of an internalstandard to the solvent flow of the LVC system.

The experiments shown measured single cell chemistry directly from thecell culture without very little, if any, sample preparation procedureswhich could negatively perturb cells. The lack of time-consuming andcostly sample pre-treatment protocols significantly improves the easeand overall speed of single cell experiments and enables the potentialof in situ chemical profiling of raw samples (e.g., water samples).Nearly any cell suspension is amenable to analysis by the invention,including mammalian cells. Targeted mammalian cell analysisidentifications are also possible. Currently, media should haveviscosities similar to water to be reliably ejected. Also, there is asmall chance (<5%) that multiple cells could be contained in the samedroplet, but those are rare events and most of them can be identified byadditional post-analysis of the images generated by the SCP. Ejection ofsmaller cells than those used herein (e.g., bacteria) is possible butwill depend on LVC-MS sensitivity to molecules of interest. Theinvention has the potential to elucidate fundamental cellular processesthat are otherwise obfuscated by removing cells from their nativeenvironment. Short sample-to-answer time, and the combination ofsingle-use dispensing cartridges with a self-cleaning LVC probe makesthe invention potentially viable for future clinical applications, andmay provide insights into how non-genetic heterogeneity of cellularmetabolites and lipids influence the efficacy of therapeutics, leadingpossibly to improved therapeutic strategies.

The invention allows for a number of unique experiments to be performed.The removal of extensive sample preparation procedures which cannegatively perturb cells make this single cell characterizationcapability much more accessible. From a single cell several mass spectracan be acquired in both an untargeted and targeted manner. Allexperiments shown herein measured single cell chemistry directly fromthe cell culture. Video images of the cell before and after dropletejection are used to calculate cell diameter and to validate that singlecells are predominantly ejected and acquired by the system. The abilityof the system to quantitate lipid peak areas at the single cell levelthrough the incorporation of an internal standard to the solvent flow ofthe system was demonstrated. Quantitative values were validated bycomparing the average of single cell measurements to that measured frombulk lipid extracts. While the targeted lipid had a <15% difference invalues between bulk lipid extracts, other lipids could be quantitated aswell including lipids outside of the internal standard class, thoughthis needs to be evaluated on a case by case basis.

In comparison to flow cytometry and mass cytometry instrumentation, theinvention offers untargeted chemical profiling of cellular metabolitesfrom unperturbed and un-modified cells. While flow cytometry isgenerally limited to one or a few molecular tags and mass cytometry toseveral dozen, the invention could incorporate potentially hundreds ofmolecular tags. Further, it is not limited to the use of fluorophores orto metal isotopes, though either could also be used sensitivitypermitting. While throughput is significantly slower as compared toconventional flow cytometry techniques, the data presented hererepresents a substantive increase over the current state of the art. Thelack of time-consuming and costly sample pre-treatment protocolssignificantly reduces the throughput of each experiment and improves theaccessibility. Single cell data could be acquired up to a theoreticalrate of 1 cell/s, in practice this was ˜1 cell/2.5 s.

Nearly any cell suspension is amenable to analysis by this technique,including mammalian cells. While the focus has been to establish theproof of concept of the SCP-LVC-MS system, targeted mammalian cellstudies are feasible potentially from human samples. One limitation ofthe technology is the droplet ejection mechanism. Currently, media musthave viscosities similar to water to be reliably ejected. Also, there isa small chance that multiple cells could be contained in the samedroplet due to dark edges along the sample cartridge. The smallest cellsize is dependent on the orifice of the sample cartridge, ability tovisualize the single cell in the sample cartridge and the massspectrometer sensitivity to the molecules of interest.

The invention as shown in the drawings and described in detail hereindiscloses arrangements of elements of particular construction andconfiguration for illustrating preferred embodiments of structure andmethod of operation of the present invention. It is to be understoodhowever, that elements of different construction and configuration andother arrangements thereof, and methods of operation other than thoseillustrated and described may be employed in accordance with the spiritof the invention, and such changes, alterations and modifications aswould occur to those skilled in the art are considered to be within thescope of this invention as broadly defined in the appended claims. Inaddition, it is to be understood that the phraseology and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

1. A method for analyzing single cells by mass spectrometry from asample containing a plurality of cells, comprising the steps of:providing a plurality of cells and a liquid medium; placing the cellsand liquid medium in a single cell isolation and ejection system;releasing liquid medium containing a single cell from the single cellisolation and ejection system; capturing the liquid medium and singlecell in a capture probe containing a flowing capture probe solvent, thecapture probe comprising a coaxial probe wherein the capture probesolvent flows from a coaxial supply flow path, overflows a common openend of the coaxial probe and enters a coaxial solvent exhaust flow path;lysing the cell with a lysis inducer in the capture probe to dispersesingle cell components into the medium; transporting the lysed singlecell components to a mass spectrometer, where the lysed single cellcomponents entering the mass spectrometer are spatially and temporallyseparated from any dispersed components of another single cell from thesample entering the mass spectrometer; and, conducting mass spectrometryon the lysed single cell components.
 2. (canceled)
 3. (canceled)
 4. Themethod of claim 1, wherein the lysis is induced by a chemical lysingagent.
 5. The method of claim 4, wherein the chemical lysing agentcomprises at least one selected from the group consisting of sodiumdodecyl sulfate (SDS), Triton-X, NP-40 lysis buffer, RIPA lysis buffer,Tween lysis buffer, CHAPS lysis buffer, and perfluorooctanoic acid. 6.The method of claim 1, wherein the lysis is induced by a liquid vortexformed by the flowing capture probe solvent.
 7. The method of claim 6,wherein the liquid vortex fluid rate is 50-300 μL/min and has a 0.5-20second elution time.
 8. (canceled)
 9. The method of claim 1, wherein thelysis is induced by the application of 0.2-1.5 volts across the cell for1-10000 μs to the cell in the capture probe solvent.
 10. The method ofclaim 1, wherein the lysis is induced by an acoustic wave emitted at thecell in the capture probe having a frequency of 15-20 kHz for 0.1-10seconds.
 11. The method of claim 1, wherein the release rate of thesingle cell isolation and ejection system is from 0.1 cell/s to 100cells/s.
 12. The method of claim 1, wherein the liquid medium comprisesa cell culture medium.
 13. The method of claim 1, wherein the releaserate of the single cell isolation and ejection system is drop on demand.14. A system for analyzing single cells by mass spectrometry,comprising: a single cell isolation and ejection system including astorage container for sample including a plurality of cells in a liquidmedium; a capture probe comprising at least one solvent supply conduitfor flowing a capture probe solvent, a capture probe exhaust conduit forreceiving single cells released by the single cell isolation andejection system and conducting lysed cell components and capture probesolvent to a mass spectrometer, and a lysis inducer, the capture probecomprising a coaxial probe wherein the capture probe solvent flows fromthe solvent supply conduit, overflows a common open end of the coaxialprobe and enters the capture probe exhaust conduit.
 15. The system ofclaim 14, further comprising a mass spectrometer having an inletconnected to the capture probe exhaust conduit.
 16. The system of claim14, wherein the lysis inducer comprises a capture probe solvent, whereinthe capture probe solvent provides a lower partial pressure than theinternal pressure of the cell to cause disruption of the cell.
 17. Thesystem of claim 16, wherein the solvent comprises at least one selectedfrom the group consisting of methanol, ethanol, isopropranol, hexane,chloroform, dichloromethane, acetonitrile, and water.
 18. The system ofclaim 14, wherein the lysis inducer comprises a liquid vortex formed bythe flowing capture probe solvent through the capture probe. 19.(canceled)
 20. The system of claim 14, wherein the lysis inducercomprises electrodes arranged on two sides of the sampling capillary forapplying a high voltage to cells in the capture probe.
 21. The system ofclaim 14, wherein the lysis inducer comprises a chemical lysing agentinjector.
 22. The system of claim 14, wherein the lysis inducercomprises an acoustic wave generator located around the outside of thesampling capillary for applying an acoustic wave to cells in the captureprobe.
 23. The system of claim 14, wherein the release rate of thesingle cell isolation and ejection system is from 0.1 cell/s to 100cells/s.
 24. The system of claim 14, wherein the release rate of thesingle cell isolation and ejection system is drop on demand.